Abstract
The spectrophotometric method of antioxidants determination using recombinant laccase Polyporus pinsitus (rPpL) and Myceliophthora ther-
mophila (rMtL) was developed. The method includes simultaneous oxidation of the antioxidant and high reactive laccase substrate producing
chromophoric radical cation. As laccase substrates ABTS and other high reactive phenoxazine derivatives: 2-phenoxazin-10-yl-ethanol (PET),
3-phenoxazin-10-yl-propane-1-sulfonic acid (PPSA) and 3-phenoxazin-10-yl-propionic acid (PPA) were used. The kinetic data were analysed
using a scheme of simultaneous oxidation of the antioxidant and the substrate.
In a range of (0.9–7.3) × 10−6 M of Trolox the measurings recovered 91 and 99% of the antioxidant if ABTS and both laccases were used. The
recovery varied between 82 and 124% if phenoxazine derivatives were used. The antioxidant activity determined in rich with antioxidants food
samples, i.e. date-palm, black raisin, golden raisin, skin of red grape, dice of red grape, fitted the literature data.
© 2006 Published by Elsevier B.V.
Please cite this article in press as: J. Kulys, I. Bratkovskaja, Talanta (2006), doi:10.1016/j.talanta.2006.11.011
+Model
TAL-8837; No. of Pages 6 ARTICLE IN PRESS
2 J. Kulys, I. Bratkovskaja / Talanta xxx (2006) xxx–xxx
Please cite this article in press as: J. Kulys, I. Bratkovskaja, Talanta (2006), doi:10.1016/j.talanta.2006.11.011
+Model
TAL-8837; No. of Pages 6 ARTICLE IN PRESS
J. Kulys, I. Bratkovskaja / Talanta xxx (2006) xxx–xxx 3
The radical cations of ABTS, PET, PPA and PPSA produced Fig. 3. The titration of radical cation of ABTS (), PET (), PPA () and
in presence of peroxidase showed strong absorbance in visible PPSA () with Trolox at pH 5.5.
area of spectrum (Fig. 2).
The radical cations of phenoxazine derivatives (PET, PPA
also may origin this difference. This inconsistency was simply
and PPSA) demonstrated remarkable stability during weeks in
eliminated by calibration with Trolox during TE determination
water solution. The addition of Trolox solution to the solu-
of real samples.
tion of the radical cation followed immediate decrease of
The generation of the radical cation of ABTS in pres-
absorbance in visible area of spectrum. The amount of consumed
ence of 1 nM of rPpL is shown in Fig. 4. The addition
radical cation was linearly proportional to the added Trolox
of Trolox stimulated the appearance of lag-period of the
(Fig. 3).
The calculated consumption of the radical cation of ABTS
Table 1
corresponded almost to 2 mol per 1 mol of Trolox since the slope The parameters of radical cations titration with Trolox at pH 5.5
of linear dependence was 1.91 ± 0.07 (Table 1). The stoichiom-
Radical cation Slope Standard error Correlation coefficient
etry of reaction for radical cations of PET, PPA and PPSA was
more than two (Table 1). The reason of this apparent stoichiom- ABTS•+ 1.91 0.07 0.9981
etry increase is unknown. This can be explained by difference of PET•+ 2.58 0.03 0.9996
PPA•+ 2.32 0.07 0.9982
correct and used for the calculations extinction coefficients. A
PPSA•+ 2.69 0.04 0.9996
more complex reaction scheme of the radical cations with Trolox
Please cite this article in press as: J. Kulys, I. Bratkovskaja, Talanta (2006), doi:10.1016/j.talanta.2006.11.011
+Model
TAL-8837; No. of Pages 6 ARTICLE IN PRESS
4 J. Kulys, I. Bratkovskaja / Talanta xxx (2006) xxx–xxx
Please cite this article in press as: J. Kulys, I. Bratkovskaja, Talanta (2006), doi:10.1016/j.talanta.2006.11.011
+Model
TAL-8837; No. of Pages 6 ARTICLE IN PRESS
J. Kulys, I. Bratkovskaja / Talanta xxx (2006) xxx–xxx 5
Table 2
Kinetic characteristics of laccase-catalysed oxidation of high reactive substrates and Trolox in 20 mM acetate buffer solution pH 5.5 at 25 ◦ C
Substrate csubstrate (M) Laccase claccase (M) k2 (M−1 s−1 ) k3 (M−1 s−1 )
ABTS 1.1 × 10−5 rPpL 1.1 × 10−9 1.4 × 106 6.4 × 105
PET 1.3 × 10−5 rPpL 1.0 × 10−9 3.6 × 106 6.4 × 105
PPA 1.5 × 10−5 rPpL 1.0 × 10−9 2.5 × 106 6.4 × 105
PPSA 1.3 × 10−5 rPpL 1.0 × 10−9 2.0 × 106 6.4 × 105
ABTS 1.4 × 10−5 rMtL 1.0 × 10−8 1.2 × 106 2.2 × 105
PET 1.3 × 10−5 rMtL 1.0 × 10−8 4.3 × 105 2.2 × 105
PPA 1.5 × 10−5 rMtL 1.0 × 10−8 9.5 × 105 2.2 × 105
PPSA 2.3 × 10−5 rMtL 1.0 × 10−8 1.5 × 105 2.2 × 105
Table 3
Trolox determination using laccase-catalysed oxidation of ABTS, PET, PPA and PPSA in 20 mM acetate buffer solution, pH 5.5, 25 ◦ C
Substrate Laccase Trolox added (M) Trolox found (M) Recovery (mean ± S.D.) (%)
Table 4 4. Conclusions
Antioxidant activity of real samples
Sample TE per 100 g of sample The experiments performed show that laccases can be used
Date-palm (1.9 ± 0.1) × 102
for antioxidant activity determination with spectrophotometer
Black raisin (3.6 ± 0.3) × 102 in presence of high active chromophoric substrates. The use of
Golden raisin (1.0 ± 0.1) × 103 laccases expands the enzymatic method of antioxidants deter-
Skin of red grape (2.4 ± 0.1) × 103 mination, and can be used if the application of peroxidases
Dice of red grape (5.0 ± 0.5) × 103 is unfavourable. As laccase substrate commercially available
Trolox (3.6 ± 0.4) × 105
ABTS was used. New N-substituted phenoxazine derivatives
TE (mean ± S.D.) was determined using 1.1 × 10−5 M of ABTS and demonstrate versatility of the suggested method. The deter-
1.0 × 10−9 M of rPpL. mined Trolox equivalent of real samples fitted described in
Please cite this article in press as: J. Kulys, I. Bratkovskaja, Talanta (2006), doi:10.1016/j.talanta.2006.11.011
+Model
TAL-8837; No. of Pages 6 ARTICLE IN PRESS
6 J. Kulys, I. Bratkovskaja / Talanta xxx (2006) xxx–xxx
the literature for both types of substrates. The method per- [10] P. Baldrian, FEMS Microbiol. Rev. 30 (2006) 215.
mits to measure submicromole concentration of an antioxidant. [11] J. Kulys, K. Krikstopaitis, A. Ziemys, J. Biol. Inorg. Chem. 5 (2000)
The optimisation of the method may significantly increase the 333.
[12] P. Schneider, M.B. Caspersen, K. Mondorf, T. Halkier, L.K. Skov, P.R.
sensitivity. Ostergaard, K.M. Brown, St.H. Brown, F. Xu, Enzyme. Microb. Technol.
25 (1999) 502.
Acknowledgement [13] D.S. Yaver, F. Xu, E.J. Golightly, K.M. Brown, S.H. Brown, M.W. Rey, P.
Schneider, T. Halkier, K. Mondorf, H. Dalboge, Appl. Environ. Microbiol.
The research was supported by Lithuanian State Science and 62 (1996) 834.
[14] F. Xu, R.M. Berka, J.A. Wahleithner, B.A. Nelson, J.R. Shuster, S.H.
Studies Foundation, projects C-03020 and C-03048. Brown, A.E. Palmer, E.I. Solomon, Biochem. J. 334 (1998) 63.
[15] Z.S. Farhangrazi, B.R. Copeland, T. Nakayama, T. Amachi, I. Yamazaki,
References L.S. Powers, Biochemistry 33 (1994) 5647.
[16] J. Kulys, K. Kriauciunas, R. Vidziunaite, J. Mol. Catal. B: Enzym. 3 (2003)
[1] B. Halliwell, Plant Physiol. 141 (2006) 312. 79.
[2] M.D. Temple, G.G. Perrone, I.W. Dawes, Trends Cell Biol. 15 (2005) 319. [17] J. Kulys, R. Vidziunaite, R. Janciene, A. Palaima, Electroanalysis 18 (2006)
[3] D. Ivekovic, S. Milardovic, M. Roboz, B.S. Grabaric, Analyst 130 (2005) 1771.
708. [18] M. Solis-Oba, V.M. Ugalde-Saldivar, I. Gonzalez, G. Viniegra-Gonzalez,
[4] T.-W. Yu, Ch. Nam Ong, Anal. Biochem. 275 (1999) 217. J. Electroanal. Chem. 579 (2005) 59.
[5] D. Villano, M.S. Fernandez-Pachon, A.M. Troncoso, M.C. Garcia-Parrilla, [19] J. Kulys, Nonlinear Anal. Model. Contr. 1 (1997) 67.
Anal. Chim. Acta 538 (2005) 391. [20] J. Kulys, R. Vidziunaite, P. Schneider, Enzyme Microb. Technol. 32 (2003)
[6] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, 455.
Free Radical Biol. Med. 26 (1999) 1231. [21] J. Vrba, J. Hrbac, J. Ulrichova, M. Modrianski, Chem. Biol. Interac. 147
[7] J.A. Vinson, Y. Hao, X. Su, L. Zubik, J. Agri. Food Chem. 46 (1998) 3630. (2004) 35.
[8] C.C. Wang, C.Y. Chu, K.O. Chu, K.W. Choy, K.S. Khaw, M.S. Rogers, [22] H. Goldberg, O. Farver, I. Pecht, J. Biol. Chem. 255 (1980) 7353.
C.P. Pang, Clin. Chem. 50 (2004) 952. [23] G. Merenyi, J. Lind, M. Jonsson, J. Am. Chem. Soc. 115 (1993) 4945.
[9] R.T.P. Correia, P. Mccue, M.M.A. Magalhaes, G.R. Macedo, K. Shetty, J. [24] A. Prakash, Medallion Lab. Anal. Prog. 19 (2001) 1.
Food Biochem. 28 (2004) 404.
Please cite this article in press as: J. Kulys, I. Bratkovskaja, Talanta (2006), doi:10.1016/j.talanta.2006.11.011